The airway epithelial tree is sculpted in the embryo via branching morphogenesis, a process in which new daughter branches sprout laterally off a main stem (domain branching) or split from the tip of a parent branch (planar or orthogonal bifurcations). These branching events are physical by nature and occur within a dynamic mechanical environment which includes the contractility of the epithelium itself, static and phasic contractions of the surrounding airway smooth muscle, and distending transmural pressures from the presence of fluid within the lumen of the tree. Although abnormal development of the embryonic lung is frequently observed in fetuses with defects that cause mechanical alterations in these tissue compartments, the physical contributions of each that are responsible for driving the branching process are unknown. Signaling downstream of Wnts regulates airway branching and smooth muscle differentiation, and is likely responsive to mechanical alterations in the developing lung. Here, we hypothesize that the mechanical behavior of airway smooth muscle plays a central role in driving branching morphogenesis, and that both epithelial contraction and luminal fluid pressure regulate branching in part by altering signaling pathways that control smooth muscle differentiation and contractility. We will combine transgenic reporter mice with high-resolution real-time spinning disk confocal microscopy, microfluidic devices, three-dimensional traction force microscopy, and computational modeling to define how the mechanical behaviors of the airway epithelium, smooth muscle, and luminal fluid collaborate to direct branching morphogenesis. In Specific Aim 1, we will use transgenic reporter mice to determine how airway smooth muscle differentiation and contraction affect domain branching and terminal (planar and orthogonal) bifurcations of the airway epithelium. In Specific Aim 2, we will use microfluidic devices to control the transmural pressure across embryonic lung explants and define the role of transmural pressure in airway smooth muscle differentiation, epithelial branching, and mechanical signaling. In Specific Aim 3, we will characterize the contractility of the airway epithelium, quantify the forces exerted by the epithelium during branching, and determine how epithelial contractility directs mechanical signaling in the surrounding mesenchyme. This work will provide a complete mechanical portrait of the tissue compartments during morphogenesis, and define how each component contributes to the physical changes required to sculpt a new branch. We expect that the mechanical behaviors and signaling pathways revealed by this work will uncover new therapeutic options to treat fetuses and neonates who present with abnormalities in lung development.